Accepted Manuscript
Title: Effects of different pruning methods on an urban tree
species: a four-year-experiment scaling down from the whole
tree to the chloroplasts
Author: A. Fini P. Frangi M. Faoro R. Piatti G. Amoroso F.
Ferrini
PII:
S1618-8667(15)00093-X
DOI:
http://dx.doi.org/doi:10.1016/j.ufug.2015.06.011
Reference:
UFUG 25563
To appear in:
Received date:
18-9-2014
Revised date:
16-4-2015
Accepted date:
25-6-2015
Please cite this article as: Fini, A., Frangi, P., Faoro, M., Piatti, R., Amoroso, G.,
Ferrini, F.,Effects of different pruning methods on an urban tree species: a
four-year-experiment scaling down from the whole tree to the chloroplasts, Urban Forestry and
Urban Greening (2015), http://dx.doi.org/10.1016/j.ufug.2015.06.011
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Accepted Manuscript
Effects of different pruning methods on an urban tree species: a four-year-experiment scaling down 1
from the whole tree to the chloroplasts 2
A. Fini1, 4, P. Frangi2,M. Faoro2, R. Piatti2,G. Amoroso2, F. Ferrini1, 3, 4, 5 3
4
1 Department of Plant, Soil and Environmental Science – University of Florence, viale delle Idee, 30, 50019, 5
Sesto Fiorentino (FI), Italy 6
2 Centro MiRT – Fondazione Minoprio, viale Raimondi 54, 22070, Vertemate con Minoprio (CO), Italy 7
3Research Unit CLimate chAnge SyStem and Ecosystem (CLASSE) - University of Florence, Florence, Italy 8
4 LABVIVA (Laboratorio per la Ricerca nel Settore Vivaistico-Ornamentale, University of Florence). 9
5 Trees and timber Institute (IVALSA) – National Research Council, Italy 10
Corresponding author: Alessio Fini: e-mail: [email protected]; Phone: +390554574024; Fax: 11 +390554574017 12 13 14 15 Abstract 16
The aim of this work was to evaluate the effects of repeated pruning interventions using different pruning 17
methods on growth and physiology of Acer pseudoplatanus L. Trees were pruned in 2008 and 2010 18
according to widely used pruning techniques for urban trees, such as reduction cut, removal cut and heading 19
(topping) cut. Crown dieback, growth of the plant and of the pruned branches, leaf morphological traits and 20
leaf gas exchange were assessed during the two growing season after each pruning cycle. Topping cut (i.e. 21
the pruning treatment which suppressed the primary axis without providing a substitute) induced changes on 22
tree growth pattern (i.e. by increasing the release of adventitious watersprouts and root suckers and 23
decreasing stem diameter growth), which were not observed in the other pruning treatments. At the leaf level 24
only topping cut increased leaf area at the expense of leaf mass per area, which may contribute to explain the 25
higher occurrence of dieback on topped branches than in control and in the other pruning treatments. Also, 26
leaves on topped branches displayed higher chlorophyll content and higher activity of Calvin cycle enzymes, 27
which did not translate in higher CO assimilation. We show here that pruning method, not only its severity 28
Accepted Manuscript
(i.e. the amount of leaf area removed), modulates the morpho-physiological response of trees to pruning and 29
that maintenance of apical control and apical dominance are key issues to preserve a structurally sound tree 30
structure, as well as the long-term efficiency of the photosynthetic apparatus. 31
32
Key words: Acer pseudoplatanus, apical control, leaf gas exchange, reduction cut, removal cut, topping cut 33
34
Introduction 35
Trees growing in the urban environment require periodic pruning to provide clearance and improve view (i.e. 36
trees along roadsides), to reduce conflicts with buildings and infrastructures, to thin dense canopies and 37
decrease wind resistance, and to reduce risk of failure by removing structural defects (Dureya et al., 1996; 38
Smiley and Kane, 2006). 39
Three types of pruning cuts are commonly used to prune urban trees (American National Standard Institute, 40
2008; Gilman, 2012): removal cut, reduction cut and heading (topping) cut. Removal cut removes the whole 41
target branch at its attachment to the trunk or parent branch, thus eliminating the entire lateral growing axis. 42
Reduction cut shortens the primary axis by removing the distal end to a smaller lateral branch, which should 43
be at least one-third to one-half of the diameter of the removed portion and should assume the apical role for 44
the remaining branch (Harris et al., 2004; Grabosky and Gilman, 2007). Finally, topping cut shortens the 45
primary axis by cutting the distal portion of the branch in the internode or in between consecutive lateral 46
branches. In this case, no properly-sized lateral is retained to assume the role of apical growing axis for the 47
remaining branch (Harris et al., 2004). These pruning methods differ in the way the target branch and its 48
apical portion are managed. It is long known that apical buds (of the tree main stem and of individual 49
branches) control key physiological processes determining tree structure and growing pattern (Cline 1994, 50
1996). These include apical dominance (i.e. the inhibition of lateral bud sprouting by the apex in an 51
individual branch) and apical control (i.e. the influence of apical growing axis on elongation and orientation 52
of lateral shoots within an individual branch) (Martin, 1987; Cline, 1997). Much of research on pruning of 53
urban trees, however, has focused on pruning severity and timing (Mierowska et al., 2002; Gilman and 54
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Grabosky, 2009; Fini et al., 2013), on tree response to wounding (Solomon and Blum, 1977; Neely, 1979; 55
Schwarze, 2008), on compartimentalization of wood decay fungi (Shigo and Marx, 1977; Schwarze, 2001; 56
O’Hara, 2007; Schwarze et al., 2007) or on tree response in the wind (Gilman et al., 2008a, 2008b; Pavlis et 57
al., 2008), whereas very little research has focused on the effects of pruning method on the long-term 58
structure and physiology of urban trees (Clark and Matheny, 2010). Because of the lack of knowledge about 59
the long-term physiological effects of pruning, it is not possible to set national and international standards 60
and best practices aimed at improving tree health and longevity and, in several countries, pruning 61
prescriptions are mostly based on operational needs and short-term cost criteria (Campanella et al., 2009; 62
Maurin and DesRochers, 2013). 63
Most of the research investigating physiological and growth response to pruning has been conducted on fruit 64
or timber trees (Lebon et al., 2004; Spann et al., 2008; Fumey et al., 2011; Maurin and DesRochers, 2013), 65
but these findings may not be directly transferred to urban trees because pruning aims are completely 66
different [i.e. improving fruit yield or quality and producing clearwood for fruit and timber production, 67
respectively, while urban arboriculture is primary targeted to obtain large, healthy, long-lived trees with a 68
sound structure, capable of providing large benefits to the community, see Nowak et al. (2002)]. Research on 69
fruit and timber plantations showed that pruning stimulates emission of new sprouts from latent and 70
adventitious buds on the pruned branch, depresses plant height and stem diameter growth, and depletes non-71
structural carbohydrates pool (Davidson and Remhprey, 1994; Spann et al., 2008), but the implications of 72
these morphological changes to long-term structural soundness were beyond the aims of these works. 73
Photosynthesis is also affected by pruning, often showing temporary increases (the so called “compensatory 74
photosynthesis”), the extent of this increase being usually related to the amount of leaf area removed 75
(Pinkard and Beadle, 1998; Medhurst et al., 2006). Whether the increase in photosynthesis is related to 76
increased leaf nutrients and chlorophyll, to higher carboxylation efficiency and ribulose regeneration, to 77
higher stomatal conductance, to the depletion of nonstructural carbohydrates pool or to altered source:sink 78
ratio is still a matter of debate (Pinkard et al., 1998; Lavigne et al., 2001; Li et al., 2002; Turnbull et al., 79
2007). 80
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The aim of this work was to evaluate the long-term effects of different pruning methods on the structure of 81
the whole tree and of the pruned branches, as well as the effects on selected leaf traits and leaf gas exchange. 82
We hypothesized that pruning method, not only its severity (i.e. the amount of leaf area removed, see 83
Pinkard and Beadle, 1998; Medhurst et al., 2006), can modulate tree response, and that greater reaction to 84
pruning by the tree may occur in treatments which mostly suppress apical control and dominance. In detail, 85
we tested the following hypotheses: 1) topping cut may completely impair apical control and dominance, 86
thereby promoting release of lateral sprouts from latent or adventitious buds and increasing the occurrence of 87
codominant branching on the pruned branches; 2) reduction cut may, instead, preserve apical dominance and 88
control, thereby resulting in much lower disturbance to tree structure; 3) the removal of the whole branch to 89
its attachment to the trunk will provide minimal disturbance to tree morphological and physiological 90
processes, because regrowth may be avoided by apical dominance exerted by the trunk; 4) all pruning 91
treatments will induce similar increases in leaf biochemical parameters and photosynthetic rate, but 92
competition among codominant sprouts will result, in the long-term, in greater decline of photosynthesis in 93
topped trees; 5) pruning effects on trees will increase as pruning is repeated over time. 94
95
Materials and Methods 96
Plant material and environmental conditions 97
In spring 2005, 28 uniform 3.2-3.8 cm diameter (10-12 cm circumference) sycamore maples (Acer 98
pseudoplatanus L.) were planted in an experimental plot at the Fondazione Minoprio (Vertemate con 99
Minoprio, Como, Italy; 45°44’ N, 9°04’ E), in a loamy sand, well drained soil. Mean annual rainfall in the 100
experimental site, calculated over the last 20 years, is 1086 mm and average temperature 12.3 °C. Daily 101
temperature and rainfall were recorded using a weather station (Vantage Pro 2, Davis, San Francisco, CA, 102
U.S.) throughout the experimental period (monthly average temperature and total rainfall are reported in Fig. 103
1). Mean yearly rainfall recorded during the experiment was greatly above the 20-year average except for 104
2011 (867 mm total rainfall), whereas mean yearly temperature was close to the 20-year average throughout 105
the experiment. 106
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Pruning treatments and experimental set up 107
After planting, trees were allowed to establish and grow undisturbed for 3 years. In February 2008 (1st 108
pruning cycle), plants were pruned using bypass hand pruners, according to the following treatments 109
(illustrated in Fig. 2): 1) Topping cut: pruning cuts were made in the middle of the internode of first-order 110
lateral branches (over 3-year old); 2) Removal cut: first-order lateral branches (over 3-year old) were cut at 111
their union with the stem, using care not to damage branch collar (Shigo, 1990); 3) Reduction cut: first-order 112
lateral branches (over 3-year old) were cut back to a lateral with sufficient size to become a new leader. 113
Therefore, all new leaders chosen had aspect ratio (calculated as ratio between the diameter of lateral chosen 114
as new leader and that of the parent branch to be removed, both measured above the branch union) greater 115
than 0.33 (Gilman, 2012); 4) Control: plants were left unpruned. In February 2010 (2nd pruning cycle), trees 116
were pruned again according to the same treatments as in 2008. All cuts were made at nodes or internodes 117
which were over 2-year old. Following the recommendations by ANSI A300 (American National Standard 118
Institute, 2008), regardless of pruning method, pruning was carried out in order to reduce leaf area by 30%, 119
which corresponds to a mild defoliation (Willard and McKell, 1978; Simard et al., 2012). Because trees were 120
pruned during the dormant season, branch cross sectional area was used to estimate the amount of leaf area 121
removed (Grabosky et al., 2007; Gilman and Grabosky, 2009). Also, while pruning, pruned material was 122
weighed in order to confirm the removal of a similar amount of woody biomass in all pruning treatments. 123
The weight of the pruned material was 1438±355 g and 2088±492 g, in the first and in the second pruning 124
cycles, respectively, and was not affected by pruning method (P = 0.333 and 0.393 in the first and in the 125
second pruning cycles, respectively). To remove the same amount of wood, removal cut required about 50% 126
less pruning cuts than topping cut and 35% less than reduction cut. In both pruning cycles six pruning cuts 127
per plant (42 per treatment) were marked with paint to be recognizable for subsequent measurements. In 128
control trees, six imaginary cuts were drawn on first-order lateral branches, similar in size and age as those 129
used in pruned treatments. Imaginary cuts were drawn next to a lateral having aspect ratio greater than 0.33 130
compared to the parent branch. In treatments where the apical bud of the branch was retained (i.e. control) or 131
substituted (i.e. reduction), the shoot bearing that apical bud was considered the leader of the branch. In 132
treatments which suppressed the apical bud (i.e. topping and removal cuts), the longest (after the first 133
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growing season) of the newly developed sprouts was considered as the new branch leader, while the 134
remaining were considered as laterals (Fig. 2). 135
136
Biometric measurements, wound closure and breaking stress 137
In both pruning cycles, all biometric parameters and wound closure were measured at the time of pruning, 138
and 12 and 24 months after pruning. Wound closure was estimated using the woundwood coefficient 139
(Scwharze, 2008), WC = 100 – [(π/4) * bt1 * ht1*100] / [(π/2) * (rt0)2], where: bt1 and ht1 are the width and the 140
height of the wound at the time of measurement, and rt0 is the initial radius of the pruning wound. 141
Stem diameter was measured at 1.3 m and stem Relative Growth Rate (RGRstem) was calculated as [ln(Øt1) - 142
ln (Øt0)] * (t1- t0)-1 where: Ø is stem diameter at times 0 and 1, and t1- t0 is time (in days) between 143
measurements (Newbery et al., 2011). The number of root suckers developed was counted in each replicate 144
tree. Then, the relative frequencies were calculated, in each treatment, as the ratio between trees releasing a 145
certain number of root suckers (i.e. 0, 1 to 4, 5 to 7, more than 7) over total number of trees of that treatment. 146
Twelve and twenty-four months after each pruning cycle, slenderness of the whole branch (L:Dwb) was 147
calculated as the ratio between the length and base diameter of pruned branches. Branch length was 148
measured from its attachment to the trunk to the apical bud, while the base diameter was measured at the 149
union with the trunk. Crown dieback was assessed visually 6 and 17 months after each pruning cycle. Crown 150
dieback frequency was calculated, in each replicate tree, as the ratio between pruned branches showing 151
dieback symptoms (i.e. extensive leaf necrosis, absence of growth, bud death) over the total number of 152
marked branches of that treatment. A pruned branch was counted for dieback if showing any of the above 153
mentioned signs on any part of the branch, including sprouts released after pruning. 154
The number, base diameter and length of watersprouts developed or released within 20 cm (as suggested by 155
Grabosky and Gilman, 2007) from the pruning cut or at the callus dieback line were measured 12 and 24 156
months after pruning. Within each marked pruned branch, the slenderness of the leader (L:Dleader) was 157
calculated as the ratio between the leader length and base diameter. Similarly, the slenderness of the lateral 158
shoots/sprouts (L:Dlateral) was calculated as the average of the slenderness of all individual sprouts 159
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(excluding, in topping and removal, the sprout designated as new leader) released from the pruning cut. 160
Length of the leader and lateral shoots/sprouts was measured from their attachment on the higher order 161
branch (in most cases, from the pruning cut) to the apical bud, while diameter was measured just above the 162
attachment. The aspect ratio between the lateral and the leader was calculated as the ratio between the base 163
diameter of each lateral shoot and base diameter of the leader. 164
The stress (σ) required to cause the failure of the attachment between the leader of the branch (or the selected 165
lateral, in control trees) and the parent branch was measured using the methods proposed by Kane et al. 166
(2008). Twenty-four months after each pruning cycle, 14 branch unions per treatment (56 in total in each 167
cycle) were attached to a dynamometer (HCB 200, Kern and Sohn Gmbh, Balingen, Germany), loaded at a 168
rate of 5 cm per minute until breakage of the attachment. Breaking stress (σ) was then calculated as: 32 * P * 169
L * sinα / (π * d3) where: P (kN) is the maximum load; L (m) is the distance between the point of application 170
of the load and the attachment which was kept fixed (about 5 cm); d (m) is the inside-bark branch diameter; 171
α (rad) is the angle between the longitudinal axis of the branch and the applied load. 172
173
Leaf gas exchange and integrated leaf anatomical traits 174
Five and seventeen months after each pruning cycle, after leaves had reached their final size, 10 fully 175
expanded leaves per tree (70 leaves per treatment) were harvested from the leader shoot/sprout of pruned 176
branches and immediately scanned using an A-3 scanner. An image analysis software (Image Tool v1.3, 177
University of Texas, San Antonio, TX, U.S.) was used to measure average leaf area. Leaves were then oven-178
dried at 70°C until constant weight to determine dry mass. Then, leaf mass per area (LMA) was calculated as 179
the ratio between leaf dry mass and leaf area. Leaf greenness index, a parameter highly correlated to leaf 180
total chlorophyll content in Acer pseudoplatanus (R2=0.9295, see Percival et al., 2008), was measured using 181
a SPAD-meter (SPAD 502. Minolta, Osaka, Japan) on the same leaves used for leaf gas exchange 182
measurements. Leaf gas exchange was measured 4, 5, 6, 15, and 17 months after the first pruning cycle, and 183
3, 5, 7, 15, and 17 months after the second pruning cycle using an infrared gas analyzer (Ciras 2, PP-System, 184
Amesbury, MA, U.S.). Measurements were conducted between 09.30 A.M. and 12.30 P.M. on the first fully 185
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expanded leaf developed on marked pruned branches (21 leaves per treatment). Leaves in the cuvette were 186
provided with saturating irradiance (1300 μmol m-2 s-1, provided using the integrated LED light unit), 187
ambient temperature, relative humidity = 60-80% air, and CO2 concentration = 380 ppm. Measured 188
parameters were: CO2 assimilation (A), stomatal conductance (gs), transpiration (E), and CO2 concentration 189
in the substomatal chamber (Ci). Instantaneous water use efficiency (WUE) was calculated as A/E. Leaf 190
temperature (Tleaf) was measured using the temperature probe integrated in the Ciras cuvette. 191
CO2 assimilation was also measured as a function of internal CO2 concentration (A/Ci curves). A/Ci curves 192
were drawn by decreasing stepwise external CO2 concentration (Ca) from 380 ppm to 30 ppm, then a Ca of 193
380 was restored and, finally, Ca was increased stepwise to 1800 ppm (Fini et al., 2014). Curves were drawn 194
3 and 7 months after the second pruning cycle (May and September, respectively). Estimates of the apparent 195
maximum rate of carboxylation by Rubisco (Vc,max) and the apparent maximum electron transport rate 196
contributing to ribulose 1,5-BP regeneration (Jmax) were made from A/Ci curves using the equations found by 197
Sharkey et al. (2007), as described in a previous work (Fini et al., 2011). The stomatal (Ls) and non-stomatal 198
limitations (Lns) to CO2 assimilation were calculated from A/Ci curves as described in Lawlor (2002) and 199
Long and Bernacchi (2003). Leaf dark respiration was calculated after 20 minutes acclimation to the 200
darkness (provided by switching off the Ciras-2 integrated light source) (Ribas-Carbo et al., 2010). Then, 201
metabolic efficiency of the leaf was calculated as A/Rdark. 202
203
Statistics 204
The experimental design was a one-tree per replicate complete randomized design with seven replicates. All 205
data were analyzed with One-Way ANOVA after checking normal distribution of data using the Shapiro-206
Wilk test (Shapiro and Wilk, 1965). Data which were not normally distributed and parameters with 207
unbalanced samples (i.e. biometrics of watersprouts) were analyzed using the non-parametric Kruskal-Wallis 208
test and means were separated using the Bonferroni test. Frequencies were calculated within each replicate 209
tree and, prior to statistical analysis, were transformed using the formula: arcsin √x, where x is the relative 210
frequency (Amoroso et al., 2010). Differences were considered significant at P < 0.05 (*) and highly 211
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significant at P < 0.01 (**). All data were analyzed using SPSS statistical software (SPSS v.20, IBM,
212 NY, U.S.). 213 214 Results 215
Effect on the whole tree and wound closure 216
Removal cuts yielded larger wounds than topping and reduction cuts, both in the first and in the second 217
pruning cycle (Table 1). Despite being small in size, wounds originated from topping cuts were the slowest 218
in closing and showed little callus and woundwood formation in the two years after pruning. 219
Before the first pruning cycle (February 2008), all trees had similar stem diameter (Table 1). Among the 220
pruning methods investigated, only topping cut depressed stem diameter growth (expressed as stem diameter 221
relative growth rate, RGRs) compared to control trees (Table 1). RGRs of topped trees was 21% and 34% 222
lower than for control trees after the first and second pruning cycles, respectively. On the contrary, neither 223
reduction nor removal treatments depressed stem growth as compared to control. 224
Presence of dieback on pruned branches increased due to topping (Table 1). In the first pruning cycle, the 225
only treatment to exhibit significant dieback was topping, which displayed dieback on 26% of pruned 226
branches (Table 1). For the second pruning cycle, dieback displayed for topping cuts (37%) and reduction 227
cuts (18%) were 4-fold and 2-fold more frequent than in control branches (9%). 228
70% of topped trees released root suckers, while only 40% of trees pruned with reduction cut, removal cut or 229
left unpruned released root suckers during the growing season after pruning. Furthermore, the frequency of 230
trees with more than 7 root suckers was greatly increased in topped plants compared to all other treatments 231
(Fig. 3A). 232
233
Effect on the pruned branches: whole branch biometrics and new growth pattern 234
All pruning techniques reduced the slenderness of the whole branch (L:Dwb) (Table 2). Removal cut 235
suppresses the whole branch, therefore L:Dwb was not measured for this treatment. In the long term (i.e. 24 236
months after pruning), slenderness of topped and reduced branches was similar, despite topped branches 237
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being less slender immediately after pruning (Table 2). During the growing season after pruning, the fast rise 238
of branch slenderness in topped trees was due to the production of very slender watersprouts in response to 239
pruning. Sprouts released after topping cuts were more slender than in all other pruning methods and had 240
similar or even higher slenderness than the unpruned shoots of control trees (Table 2). The increase in 241
slenderness of topped branches was due to increased primary growth, rather than decreased secondary 242
growth (data not shown, but primary growth of the leader shoot was on average 190% and 245% greater than 243
in reduction cut and control, respectively, after 12 months since pruning). 244
All types of pruning stimulated the release of watersprouts in the proximity of the wound, or directly from 245
the callus (Fig. 3B). In all treatments except topping, however, less than two watersprouts were developed in 246
the 20 cm proximal to the cut in over 80% of pruned branches. On the contrary, over 55% of topped 247
branches released up to 4 watersprouts and over 15% released five or more adventitious sprouts (Fig. 3B). In 248
both pruning cycles, the aspect ratio between the leader and lateral shoots/sprouts within 20 cm from pruning 249
cut was higher in those treatments (removal and topping cuts) which suppressed the apical shoot of the 250
branch without preserving (i.e. control) or substituting (i.e. reduction cut) it (Table 2). 251
The stress (σ) required to cause the failure of the attachment between the leader and the parent branch was, 252
on average, 64% and 36% lower in topped than in reduced and control branches in the first and in the second 253
pruning cycle, respectively (Table 2). On the contrary, σ in reduction cut and removal cut (the latter 254
measured only in the second cycle) did not differ from control. 255
256
Effect on leaf characteristics and gas exchange 257
Pruning method largely impacted leaf anatomy (Table 3). Leaves developed on topped branches were larger 258
and had lower LMA than in other pruning treatments and in control trees. In the first pruning cycle, the effect 259
of topping on leaf size and LMA was significant in the first growing season after pruning, but not in the 260
second one. As pruning was repeated, the effect of pruning method on leaf size and LMA lasted longer, and 261
leaves developed on topped branches still had larger leaf area and lower LMA even in the second growing 262
season after pruning. Leaves originated on topped branches were about 1 °C warmer than leaves of control 263
branches during the late-spring and summer period (data are the average of three measurement days 264
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conducted from May to September) (Table 3). On the contrary, neither removal nor reduction treatments lead 265
to significant leaf warming compared to control. 266
Only topping cut increased significantly the leaf greenness index (Table 3). The effect was indeed transitory, 267
being only significant in the growing season immediately after pruning, then disappearing or being greatly 268
reduced the following season. Similarly, the apparent carboxylation efficiency by Rubisco (Vc,max) and the 269
apparent contribution of electron transport to Ribulose regeneration (Jmax) were higher in the topping 270
treatment than in control during the first growing season after pruning (Table 3). Significant difference in 271
Vc,max and Jmax were found between these two treatments both in May and in September, 3 and 7 months after 272
pruning, respectively. On the contrary, leaves developed on reduced or removed branches had similar Vc,max 273
and Jmax to control. 274
The effects of pruning method on CO2 assimilation (A) were mostly restricted to the first few months 275
following pruning (i.e. 3 and 4 months after the second and the first pruning cycle, respectively) (Fig. 4A). 276
Early after pruning, only leaves of topped branches displayed higher A than control trees in both pruning 277
cycles. Later on during the growing season, differences among treatments disappeared, except on late 278
summer 2011 (17 months after the second pruning cycle), when the removal treatment displayed lower A 279
than the reduction and control treatments. Stomatal conductance (gs) was not affected by pruning method in 280
the first pruning cycle (Fig. 4B). When pruning was repeated, an early enhancement of gs was observed 3 281
months after pruning in the topping and reduction treatments compared to removal and control (Fig. 4B). 282
Later on in the growing season (i.e. 5 and 7 months after 2nd pruning cycle, July and September respectively), 283
gs decreased in topped trees and increased in control, making the differences in gs less substantial. In the 284
second growing season after the 2nd pruning cycle, leaves held on branches developed after the removal cut 285
had lower gs than leaves of the other treatments. Intercellular CO2 concentration (Ci) was generally decreased 286
by topping and removal cuts in the first summer after the first pruning cycle (5 and 7 months after pruning), 287
then differences were not confirmed in the second growing season (15 and 17 months after pruning) (Fig. 288
4C). As pruning was repeated, the lower Ci during summer in leaves of the topping treatment, compared to 289
control, was confirmed (5 and 7 months after the second pruning cycle), and differences were still significant 290
in the second growing season (15 and 17 months after pruning) (Fig. 4C). 291
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Three months after pruning, early after full leaf expansion (May 2010), all types of pruning reduced stomatal 292
limitation to CO2 assimilation (Ls) compared to control (Table 4). Similarly, non-stomatal limitation 293
(including mesophyll diffusion and biochemical limitations) (Lns) were lower in pruned than in control 294
leaves. In detail, topping cut lead to the largest decrease in Lns, whereas removal cut the least (Table 4). As 295
season progressed, and trees had to cope with stresses such as heat and reduced water availability (see the 296
change in air temperature and rainfall from May to July 2010 in fig. 1), Ls increased to a greater extent in 297
topping than in removal and reduction treatments, while control showed the lowest increase (Table 4, 7 298
months after pruning). Similarly, Lns increased in all pruned treatments, but particularly in leaves of topped 299
trees which, however, yet displayed lower Lns than control trees, as shown by the negative Lns value. 300
301
Discussion 302
Shigo (1989) described pruning as “the best thing an arborist can do for a tree but at the same time, one of 303
the worse things an arborist can do to a tree; much depends on how pruning is carried out”. Results of this 304
experiment support Shigo’s statement by providing a quantitative evaluation of the effects of different 305
pruning methods, scaling down from the whole tree to leaf physiology and biochemistry. 306
Pruning treatments mainly differed because the apical bud of the pruned branch was suppressed (topping), 307
substituted (reduction) or retained (control), while removal cut suppressed the whole primary branch, instead 308
of its apical portion. We hypothesized that these methods may differently disturb apical dominance thereby 309
affecting subsequent growth and physiological processes and, in particular, that substituting the apical bud of 310
the branch with the one of a properly sized lateral branch through reduction cut may, at least in part, avoid 311
the complete release of apical dominance which occurs after chopping off (i.e. topping) (Hillman, 1984). 312
Results of this study clearly confirm this hypothesis. 313
First, only topped trees showed reduced stem diameter growth and increased release of root suckers 314
compared to controls. Reduction in stem diameter growth have been reported for intense pruning treatments 315
(i.e. > 50% leaf area, Pinkard and Beadle, 1998; Neilsen and Pinkard, 2003), but were unexpected here, as 316
only 30% of tree canopy was removed (Maurin and DesRochers, 2013). Because the amount of leaf area 317
removed by all pruning treatments was similar, it is unlikely that diminished stem growth of topped trees is 318
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due to reduced availability of photosynthetates. Unlike other pruning methods, topping cut most likely 319
triggered the change in biomass partitioning to favour neoformed sprouts, at least partly at expenses of stem 320
growth, as reported to occur in severely defoliated trees (Hoogesteger and Karlsson, 1992; Pinkard and 321
Beadle, 1998). This is consistent with the higher emission of root suckers and watersprouts observed in 322
topped than in control trees. Enhanced release of sprouts from lateral, adventitious and latent bud has been 323
related to suppressed apical dominance (Cline, 1997). Consistently, our data show that while topping had a 324
severe effect on tree structure by greatly suppressing apical control and promoting epicormic growth, 325
pruning back a branch to a lateral with intact apical bud and large enough to become the new branch leader 326
(i.e. reduction cut) preserved normal tree growth pattern (Wilson, 2000). Similarly, removal of the whole 327
branch at its attachment to the trunk resulted in minimal disturbance to tree structure. In fact, epicormic 328
sprouts developed next to the removal pruning cut grew in the inner part of the crown (particularly after the 329
2nd pruning cycle, when trees were larger) under reduced light availability, which greatly limit their primary 330
growth and slenderness (Solomon and Blum, 1977). This is probably due to lower sink strength than the stem 331
they are attached to, resulting in photoassimilates export from the sprouts (Stoll and Schmid, 1998; Wilson, 332
2000). One of major disturbances of pruning to tree structural strength is that it inextricably leads to open 333
wounds, which may be a preferential point of entry for wood decay fungi. In this experiment, removal cuts 334
yielded larger wounds than all other treatments but, contrary to the previously reported inverse relation 335
between wound size and time of closure (Solomon and Shigo, 1976), wounds from removal cuts were the 336
fastest at closing. Not only wound size, but also the wound location within a tree, can affect wound closure 337
process. Larger wounds have been shown to lead to greater amount of discoloured wood, while poor 338
correlations are generally found between the amount of discoloured wood and closure time (Solomon and 339
Shigo, 1976; Gilman and Grabosky, 2007). It was shown, however, that the amount of wood discoloration is 340
inversely related to the vigour of the wounded plant organ, and that wound closure time is also inversely 341
related to vigour (Solomon and Blum, 1977; Armstrong et al., 1981). 342
Second, on the pruned branches, codominance of newly developed sprouts was triggered in treatments which 343
suppressed the apical axis or the whole branch, without providing a new leader. In fact, new sprouts 344
(branches in the following year) developed on topped branches and after branch removal had aspect ratio 345
Accepted Manuscript
occurs between leader and subordinate branches (Grabosky and Gilman, 2007; Gilman, 2012). We show here 347
that if the apical branch is substituted by a properly sized lateral, the latter has enough sink strength to 348
prevent extensive outgrowth from lateral buds and to maintain apical control over subodrdinate laterals, 349
indicating that reduction cut achieves in maintaining apical control whereas topping cut does not. From a 350
management viewpoint, codominance is one of the most hazardous structural defects of a tree which usually 351
leads to reduced tree safety, particularly if codominant branches are slender and weakly attached to the trunk 352
or the parent branch (Dahle et al., 2006; Gilman, 2012; Ciftci et al., 2013). Indeed, the stress required to 353
break the union between newly developed sprouts and their parent topped branch was about 1/3 to 2/3 lower 354
than that required to tear apart a normal branch union. This corroborates the idea that, in topped branches, 355
most of regrowth occurs from adventitious buds, which are inextricably weakly attached to the parent 356
branch, since they are attached at the cambium level (Dahle et al., 2006). Also, sprouts released in topped 357
branches were more slender than in other pruning treatments and, after the second pruning cycle, the leader 358
sprouts of topped branches were even more slender than unpruned shoots in control plants, although the 359
same was not observed for lateral sprouts. High slenderness may not be an issue for young growing axis, 360
which are flexible enough to avoid fractures even at high wind loads, which may instead cause the failure of 361
the attachment, particularly if the branch union is weak (Bertram, 1989). As branches grow old and increase 362
in size, switching from a “light-harvesting” to a structural role (which occurs when the branch is about 3 m 363
in length) slenderness starts to decline (Bertram, 1989; Dahle and Grabosky, 2010), because of reduced 364
elongation, rather than to smaller diameter growth (Dahle and Grabosky, 2010). Topping cut hinders this 365
normal ageing process of the branch by stimulating primary branch growth (long about 2.7 m just before the 366
second pruning cycle) and prevents the branch from performing a structural role. Pioneer works 367
hypothesized that removal of the apical axis may stimulate lateral axis to elongate more than they would 368
have done if the terminal had remained intact (Wilson, 1990), because of altered hormonal balance (Thimann 369
and Skoog, 1934; Prochazka and Jacobs, 1984), demonstrating that all lateral shoots have the potential to 370
become long shoots if not dominated (Suzuki and Kohno, 1987). The effects of reduction, removal and 371
topping cuts on tree hormones was not tested in this experiment; however, this knowledge would be of great 372
importance for determining best pruning practices and deserves to be addressed by future research. The fast 373
growth rate (in length) of pruned branches, the increased codominant branching and the weak branch 374
Accepted Manuscript
attachments in topped branches out-compassed the safety benefit resulting from the initial greater reduction 375
of whole branch slenderness immediately after pruning. Thus, despite topping appearing as a cheap and fast 376
pruning method in the short term, it has deleterious mid- and long-term effects on tree structure, thereby 377
resulting in the need of more frequent pruning and in a 4-fold rise of overall pruning cost (Campanella et al., 378
2009). 379
Third, from the physiological viewpoint, topping stimulated vigorous resprouting from pruned branches, but 380
at the expenses of stem diameter growth and of the capacity to withstand unfavourable conditions in the 381
long-term (Harris et al., 2004; Spann et al., 2008). This may be due to the enhanced competition for light and 382
nutrients among watersprouts released from the same pruning cut. After apical control is removed by a 383
properly executed cut, the distal branch grows larger and more vertical, replaces the removed terminal and 384
restores apical control (Wilson, 2000). On the contrary, topping cut releases several co-dominant sprouts all 385
located close to the wound without any distal shoot. In this situation, becoming larger and developing larger 386
leaf area provides competitive advantage, because of higher hormone production and greater light harvesting 387
capacity compared to shorter sprouts with smaller leaf area (Wilson, 2000). Growth rate greatly depends on 388
leaf structural, biochemical and functional characteristics, with leaves with small LMA and high nitrogen 389
and chlorophyll content being commonly associated with fast-growing strategies (Reich et al., 1992: Poorter 390
and Bongers, 2006). Among pruning treatments tested here, only topping affected leaf structural traits such 391
as leaf area and leaf mass per area. The larger area of individual leaves of topped branches increased the 392
photosynthetic surface of individual branches, but resulted in leaf over-heating because of lower heat 393
dissipation by conduction/convection, than smaller leaves (Nobel, 2005). Increased leaf area in topped trees 394
was paralleled by a decrease in leaf mass per area. Leaves with low LMA are productive and often associated 395
with fast-growing plant strategies, but are necessarily short-lived and more susceptible to environmental 396
stresses (Wilson et al., 1999; Bussotti, 2008; Poorter et al., 2009). Consistently, a greater occurrence of 397
crown dieback was observed in topped trees than in other treatments after both pruning cycles. 398
Transient (lasting few weeks to few months) increases in net CO2 assimilation (compensatory 399
photosynthesis) have been reported to occur following pruning and partial defoliation, with the magnitude of 400
this increase being positively correlated with pruning/defoliation severity (Pinkard et al., 1998; Hart et al., 401
Accepted Manuscript
2000; Turnbull et al., 2007). We show here that the type of pruning, not only its severity, can modulate tree 402
responses at the leaf level. Mechanisms leading to compensatory photosynthesis are still poorly understood 403
and may involve increased stomatal conductance, increased leaf nitrogen and chlorophyll, and increase Vc,max 404
and Jmax (Sharkey, 1985; Pinkard et al., 1998; Pinkard and Beadle, 1998; Turnbull et al., 2007). Leaf 405
structure is generally optimized for maintaining the operating [CO2] in the chloroplast stroma (Cc) at the 406
transition between the Rubisco carboxylation and RuBP regeneration limitations to photosynthesis (Farquhar 407
et al., 1980), to reduce photorespiration and, consequently, increase CO2 assimilation (Terashima et al., 408
2011). A tight co-regulation of stomatal and non-stomatal factors is required to achieve this goal (Flexas and 409
Medrano, 2003). Topping cut lead to an imbalance of stomatal regulation (when compared to control) which 410
was not observed in other pruning treatments. In fact, non-stomatal limitations to photosynthesis were much 411
lower in topping than in other pruning treatments and than in control, because Rubisco activity and the 412
contribution of electron transport to ribulose regeneration were greatly up-regulated in leaves developed in 413
topped branches. Higher Vc,max and Jmax in topping treatment resulted in transient increases in net CO2 414
assimilation, when stomatal conductance was high enough to maintain adequate leaf internal CO2. Later in 415
the growing season, however, stomatal limitations increased more in leaves of topped plants than in other 416
treatments. Although leaves on topped branches still showed higher Vc,max and Jmax after the summer period 417
(7 months after 2nd pruning cycle), they did not show enhanced CO
2 assimilation rate compared to other 418
treatments because of high stomatal limitations, as previously hypothesized (Pinkard et al. 1998; Pinkard and 419
Beadle 1998). Maintaining higher Vc,max and Jmax requires large complements of enzymes and other 420
metabolites which have a substantial maintenance cost and require periodic (and costly) recycling (Reich et 421
al., 1998). Moreover, higher leaf chlorophyll and nitrogen content are commonly associated with higher 422
respiration rates (Reich et al., 1998). Consistently, the A/Rd ratio was significantly lower in leaves on topped 423
branches than in other treatments as soon as CO2 assimilation declined because of stomatal limitation, 424
indicating that metabolic inefficiency at the leaf level is promoted by topping (Cai et al., 2009). 425
The morpho-physiological changes induced by topping were not found in plants pruned by the reduction cut, 426
suggesting that apical dominance and control may be effectively retained if the branch is pruned to a lateral, 427
large enough to become the new dominant primary axis. Removal cut, similar to topping cut, removes the 428
primary axis without proving a substitution leader. However, disturbance to plant physiology was much 429
Accepted Manuscript
lower, as watersprouts developed from pruning cut grow in the inner part of the canopy, and self-shading 430
resulted in a generally low photosynthetic rate, stomatal conductance and, presumably sink strength 431
(McCormick et al., 2006). This effect was clear particularly after 2nd pruning cycle, when plants were larger 432
and with broader and denser canopies, which resulted in a denser shade cast on new shoot developing from 433
the trunk. 434
In conclusion, we show here that pruning method, not only its severity, modulates the morpho-physiological 435
response of trees to pruning. Maintenance of apical control and apical dominance are key issues to preserve a 436
structurally sound tree structure, as well as the long-term efficiency of the photosynthetic apparatus. While 437
removal of the whole primary axis at its attachment to the trunk provide minimal disturbance to tree 438
physiology, shortening of the branch may provide different results, depending on where the branch is 439
shortened. Reducing the primary axis to a lateral branch large enough to become the new branch leader 440
appeared to preserve normal branching pattern and had little effects on leaf structure and photosynthetic 441
performance. On the contrary topping a branch (shortening of the primary axis without providing a 442
substitution leader) greatly affected tree structure and functioning by altering branching pattern, by 443
promoting competition among sprouts of the same branch, and by determining a shift toward a more pioneer 444
(fast growing) behaviour, but at the expense of tolerance to environmental stresses. It must be noted, 445
however, that this work dealt with young trees and further research is needed to evaluate the physiological 446
response to pruning method in mature or senescing trees. 447
Acknowledgements 448
This work has been done as a part of a research project called “Miglioramento delle tecniche produttive e 449
della qualità del prodotto nel vivaismo ornamentale - TECPRO” financed by Regione Lombardia – 450
Agricultural Department, according to the Plan of Research and Development 2008. Partial funding was 451
provided by UNISER Consortium, Pistoia (Italy) 452
453
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Accepted Manuscript
633
Figure captions 634
Figure 1: Monthly average temperature (°C) and rainfall (mm) at the experimental site (Vertemate con 635
Minoprio, CO, Italy, 45° 44’ N, 9° 04’ E, 250 m above sea level) during the experimental period (2008 – 636
2011). 637
Figure 2: Schematic diagram of the pruning treatments imposed and of the effects of the different pruning 638
methods on new growth. The black triangles indicate that the apical bud of the branch was left untouched. 639
“Leader” and “laterals” indicate which shoots/sprouts were considered as dominant and subordinate growing 640
axes, respectively. 641
Figure 3: Frequency of: (A) number of root suckers released from the trunk flare, and (B) number of 642
watersprouts released within 20 cm from pruning cut during the first growing season after the first pruning 643
cycle. Frequencies were calculated on 7 replicate trees per treatment (root suckers) or 42 replicate pruning 644
cut per treatment (watersprouts). Different letters within the same frequency class indicate significant 645
differences among treatments at P < 0.05 (*) or P < 0.01 (**). 646
Figure 4: Effect of different pruning treatments on: A) CO2 assimilation (A, μmol m-2 s-1); B) stomatal 647
conductance (gs, mmol m-2 s-1); and C) CO2 concentration in the substomatal chamber (Ci, ppm) measured in 648
the 17 months after the first and the second pruning cycle. Different letter within each sampling date indicate 649
significant difference among treatments at P < 0.05 (*) or P < 0.01 (**). 650
651 652
Table 1: Effect of different pruning methods on wound size (cm2) and wound closure (estimated using the 653
woundwood coefficient, see method section for details), on stem relative growth rate (RGR, μm cm-1 d-1) and 654
on the frequency of dieback on pruned branches after the first and the second pruning cycle. Stem diameter 655
measured in February 2008, right before the first pruning cycle, is also reported. Different letters within the 656
same row denote significant differences among pruning treatments at P < 0.01. n.d. = not determined. 657 Pruning cycle Months after pruning
Topping Reduction Removal Control P
1 0 2.5 b 2.7 b 4.2 a n.d. 0.000
Accepted Manuscript
1 12 0 b 65 a 44 b n.d. 0.000 1 24 10 b 93 a 72 a n.d. 0.000 2 12 4 b 17 a 19 a n.d. 0.000 Woundwood coefficient (%) 2 24 24 b 43 a 50 a n.d. 0.000 Stem diameter (cm) 1 0 6.1 6.2 6.6 6.3 0.232 1 0-24 8.1 b 10.8 a 10.0 a 10.3 a 0.003 RGRstem (μm cm-1 d-1) 2 0-24 6.2 b 8.5 a 8.8 a 9.4 a 0.001 1 17 26 a 0 b 3 b 0 b 0.008 Crown dieback (%) 2 17 37 a 18 b 6 c 9 c 0.005 658 659Table 2: Effects of different pruning methods on branch biometrics: slenderness of the whole branch (L:Dwb, 660
cm cm-1); aspect ratio between the dominant and the subordinate shoots within 20 cm from pruning cut(cm 661
cm-1); slenderness of the dominant shoot/sprout of the branch (L:D
leader, cm cm-1) and of subordinate 662
shoots/sprouts (L:Dlateral, cm cm-1) and stress required to cause the failure of the union between the dominant 663
shoot of the branch and the parent branch (σ, MPa). Different letters within the same row denote significant 664
differences among pruning treatments. n.d. = not determined 665 666 Pruning cycle Months after pruning
Topping Reduction Removal Control P
1 0 24.2 c 35.4 b n.d. 63.7 a 0.000 1 12 58.5 b 64.6 b n.d. 81.5 a 0.000 1 24 75.8 b 75.9 b n.d. 85.9 a 0.004 2 0 18.3 c 57.2 b n.d. 88.4 a 0.000 2 12 46.8 c 64.4 b n.d. 89.1 a 0.000 L:Dwb (cm cm-1) 2 24 69.9 b 71.0 b n.d. 89.1 a 0.002 1 12 0.86 a 0.34 b 0.82 a 0.31 b 0.000 1 24 0.80 a 0.34 b 0.75 a 0.41 b 0.008 2 12 0.77 a 0.29 c 0.91 a 0.47 b 0.004 aspect ratio (cm cm-1) 2 24 0.78 a 0.30 b 0.73 a 0.46 b 0.009 1 24 94.2 a 79.4 b 60.5 c 89.9 a 0.008 L:Dleader (cm cm-1) 2 24 95.2 a 80.6 b 60.3 c 75.5 b 0.000 1 24 84.6 a 79.3 b 52.3 c 80.9 ab 0.015 L:Dlateral (cm cm-1) 2 24 89.9 a 71.2 b 51.3 c 86.9 a 0.000 1 24 20.1 b 47.0 a n.d. 53.7 a 0.020 σ (MPa) 2 24 37.4 b 62.4 a 47.4 ab 58.5 a 0.029 667
Table 3: Effects of different pruning methods on leaf morpho-physiological characteristics: average leaf area 668
(cm2), leaf mass per area (LMA, g m-2), leaf temperature (T
leaf, °C), leaf greenness index, apparent rate of 669
carboxylation by Rubisco (Vc, max, μmol m-2 s-1); apparent contribution of electron transport to ribulose 670
regeneration (Jmax, μmol m-2 s-1), and ratio between net CO2 assimilation and dark respiration (A/Rdark). 671
Different letters within the same row denote significant differences among pruning treatments at P < 0.05. 672
Accepted Manuscript
cycle pruning 1 5 270.8 a 199.0 b 188.0 b 220.8 b 0.000 1 17 210.1 166.2 152.5 172.1 0.683 2 5 279.9 a 165.22 b 155.3 b 147.8 b 0.010 Leaf area (cm2) 2 17 183.5 a 165.9 b 155.9 b 131.6 c 0.000 1 5 80.1 b 98.9 a 99.9 a 93.1 a 0.038 1 17 87.3 93.3 97.8 93.8 0.817 2 5 88.6 c 106.5 a 96.2 b 104.5 ab 0.016 LMA (g m-2) 2 17 78.5 b 95.8 a 93.7 a 94.4 a 0.003 1 4-6 30.5 a 29.6 b 29.3 b 29.4 b 0.000 1 15-17 29.7 a 29.3 b 29.0 b 29.2 b 0.000 2 3-8 31.0 a 30.1 b 30.0 b 29.9 b 0.000 Tleaf (° C) 2 15-17 29.3 a 28.4 b 28.6 b 28.3 b 0.000 1 3-8 45.0 a 42.9 b 39.0 c 40.2 bc 0.000 1 15-17 40.0 40.9 39.6 40.1 0.482 2 3-8 42.7 a 39.1 b 35.0 c 36.9 bc 0.000 Leaf greenness index (a.u.) 2 15-17 39.6 a 38.3 a 35.7 b 37.3 ab 0.005 2 3 124.0 a 103.2 ab 93.4 b 89.5 b 0.030 Vc,max (μmol m-2 s-1) 2 7 133.6 a 98.0b 93.1 b 96.0 b 0.000 2 3 226.3 a 165.5 b 141.4 b 130.2 b 0.001 Jmax (μmol m-2 s-1) 2 7 198.0 a 156.0 b 152.6 b 146.3 b 0.000 2 3 13.06 14.34 14.11 13.08 0.754 A/Rdark 2 7 8.91 b 13.57 a 13.72 a 12.43 a 0.000 673Table 4: Stomatal (Ls) and non-stomatal (Lns) limitations to photosynthesis in leaves of A. pseudoplatanus 674
developed on branches subjected to different pruning methods. Lns was calculated as in Lawlor (2002) and 675
Long and Bernacchi (2003) using control leaves as reference parameter. Negative Lns indicates lower non-676
stomatal limitations to CO2 assimilation than in control trees. Different letters within the same row denote 677
significant differences among pruning treatments at P < 0.05. 678 Pruning cycle Months after pruning
Topping Reduction Removal Control P
Ls (%) 2 3 9.8 b 9.9 b 10.9 b 16.6 a 0.035 2 7 41.0 a 21.9 b 21.9 b 18.8 b 0.015 Lns (%) 2 3 -52.3 c -16.6 b -2.7 a - 0.039 2 7 -25.4 b 5.4 a 4.4 a - 0.012 679 680
• Pruning method, not only its intensity, modulates the tree response to pruning 681
• Reducing the apical growing axis to a lateral little disturbs branch growth 682
• Topping increases codominance and weakens branch structure 683
• In topping, higher Vc,max and Jmax are not paralleled by higher CO2 assimilation 684
